The DNA damage response coordinates cell cycle transitions, DNA replication, DNA repair, and apoptosis to guard against genomic instability. Three related kinases belonging to the PI3K-related protein kinase (PIKKs)− family, ataxia telangiectasia-mutated (ATM), ATM and RAD3-related (ATR), and DNA-dependent protein kinase (DNA-PK), are involved in the DNA damage response. ATR is essential for viability in replicating human cells, and disruption of ATR in mice results in embryonic lethality (see, for example, Cortez et al., (2001) Science 294, 1713-1716). ATR regulates replication fork stability, restart of collapsed forks, and late-origin firing during S-phase. This kinase also activates the G2 checkpoint to prevent entry into mitosis in the presence of damaged DNA (see, for example, Sancar et al., (2004) Annu. Rev. Biochem. 73, 39-85).
Hypomorphic mutations in ATR cause Seckel syndrome and ATR mutations have been associated with poor prognosis of tumors with microsatellite instability (O'Driscoll, (2003) Nat. Genet. 33, 497-501; Zighelboim et al., (2009) J. Clin. Oncol. 27, 3091-3096: Lewis et al., Genes Chromosomes Cancer 46, 1061-1068). A key function of ATR is activation of checkpoint kinase 1 (Chk1), leading to cell cycle arrest. Many anti-cancer therapeutics induce DNA damage and ATR is itself a drug target.
ATM, ATR and DNA-PK undergo autophosphorylation. Phosphorylation sites on ATM and DNA-PK are functionally significant and have been used as direct markers of activation (see, for example, Kozlov et al., (2006) EMBO J. 25, 3504-3514). Phosphorylation at the threonine at position 1989 (T1989) is important for ATR activation (Nam et al., (2011), J. Biol. Chem. 286: 28707-28714). There is a need for reagents that can be used to detect ATR phosphorylation at this position.